Modulating CagA Expression in H. pylori Infection

The GFP-MAP1LC3B plasmid and RFP-MAP1LC3B expression plasmid were kindly provided by Dr. Tamotsu Yoshimori (Department of Cell Biology, National Institute for Basic Biology, Presto, Japan) and Dr. Maria Colombo (Universidad Nacional de Cuyo, Mendoza, Argentina), respectively. The CagA expression plasmid, pEGFP-C1-CagA (GFP-CagA) (Asahi et al., 2000 (link); Suzuki et al., 2009 (link)), was kindly provided by Dr. Chihiro Sasakawa. The cagA mutant plasmid, pEGFP-C1-CagA-Mut (GFP-CagA-Mut) was constructed by Life Technologies, Shanghai, China, and a series of CagA mutants with the Tyr residues of 899, 918, and 972 being substituted by Ala were generated from a plasmid-encoding fragment of cagA gene of H. pylori ATCC 26695 on pBluescript (Promega, Madison, USA) using a Gene Editor in vitro Site-Directed Mutagenesis System (Promega). These mutants were at the sites 2,695–2,697, 2,752–2,754, and 2,914–2,915 bp, respectively, started from the sequence ATG. Lipofectamine 2000 (Invitrogen, #11668019) was used to transfect plasmids and/or siRNAs into AGS cells. 3 × 10⁶ AGS cells were seeded into a 100-mm dish and incubated with transfection complexes containing 100 nM siRNA for 24 h.